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Biochemistry 1984, 23, 5467-547 1 Pedersen, S . E., & Ross, E. M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7228-7232. Rimon, G., Hanski, E., & Levitzki, A. (1980) Biochemistry 19, 4451-4460. Ross, E. M., & Gilman, A. G. (1980) Annu. Rev. Biochem. 49, 553-564. Schaffner, W., & Weismann, C. (1973) Anal. Biochem. 56, 502-514. Shorr, R. G. L., Lefkowitz, R. J., & Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826. Shorr, R. G. L., Strohsacker, M. W., Lavin, T. N., Lefkowitz, R. J., & Caron, M. G. (1982) J. Biol. Chem. 257,
12341-1 2350. Smigel, M. D., Northup, J. K., & Gilman, A. G. (1982) Recent Prog. Horm. Res. 38, 601-622. Smigel, M. D., Ross, E. M., & Gilman, A. G. (1984) in Cell Membranes, Methods and Reviews (Frazier, W., Elson, E., & Glaser, L., Eds.) Chapter 7, pp 274-294, Plenum Press, New York. Sternweis, P. C., Northup, J. K., Smigel, M. D., & Gilman, A. G. (1981) J . Biol. Chem. 256, 11517-11526. Tolkovsky, A. M., & Levitzki, A. (1978) Biochemistry 17, 3795-3810.
Catecholamine-Stimulated Guanosine 5 / 4 4 3-Thiotriphosphate) Binding to the Stimulatory GTP-Binding Protein of Adenylate Cyclase: Kinetic Analysis in Reconstituted Phospholipid Vesiclest Tomiko Asanot and Elliott M. Ross*
ABSTRACT: The stimulatory GTP-binding protein of adenylate cyclase, G,, and fl-adrenergic receptors were reconstituted into unilamellar phospholipid vesicles. The kinetics of the quasiirreversible binding of guanosine 5’-0-(3-thiotriphosphate) (GTPyS) to G,, equivalent to G, activation by nucleotide, was studied with respect to the stimulation of this process by padrenergic agonists and Mg2+. The rate of GTPyS binding displayed apparent first-order kinetics over a wide range of nucleotide, agonist, and Mg2+concentrations. In the absence of agonist, the apparent first-order rate constant, kapp,was 0.17-0.34 min-’ and did not vary significantly with the concentration of nucleotide. At 50 mM MgC12, kappincreased somewhat, to 0.26-0.41 min-’, and remained invariant with
the nucleotide concentration. In the presence of agonist, kapp was dependent on nucleotide concentration. At M GTPyS, the addition of (-)-isoproterenol caused at most a 2-fold stimulation of kaqp. However, kappmeasured in the presence of isoproterenol increased as an apparently saturable function of the GTPyS concentration, such that isoproterenol caused a 17-fold increase in kappat 1 pM GTPyS. The effect of isoproterenol on kappalso appeared to saturate at high isoproterenol concentration, yielding a kapp 6 min-‘ at high concentrations of both nucleotide and agonist. These data suggest that the receptor-agonist complex acts by increasing the rate of conversion of a lower affinity G,-GTPyS complex to the stable activated state.
T e activity of hormone-sensitive adenylate cyclase primarily reflects the extent of activation of the stimulatory GTP-binding protein, G,.’ Activation of G, occurs upon the high-affinity binding of GTP, or a GTP analogue such as GTPyS, and is manifest as the ability of nucleotide-ligandedG, to bind to and stimulate the catalytic unit of adenylate cyclase. Activation is terminated either by the slow dissociation of nucleotide or, in the physiological case, by the hydrolysis of GTP to GDP. GDP does not activate G,. Receptors for stimulatory hormones function by increasing the rate of activation of G, by guanine nucleotides. This increase in the rate of activation increases the steady-state concentration of G,-GTP or, in the case of nonhydrolyzed analogues, increases the rate at which the active G,-nucleotide complex is formed [reviewed by Ross & Gilman (1980) and Smigel et al. (1984)l.
The mechanism of activation of G, by nucleotides has been addressed most thoroughly by Gilman’s group using purified, detergent-solubilized G, (Northup et al., 1982, 1983; Smigel et al., 1982). G, is a dimer of a 45 000-Da (or 52000-Da) GTP-binding a subunit and a 35 000-Da subunit.2 These authors showed that the free a subunit-nucleotide complex is the active form of G,. GTPyS binds to G, and activates it by a slow reaction that is tighly coupled to the dissociation of the 35 000-dalton p subunit. The binding reaction is first order in G,, and the observed first-order rate constant for binding, kapp,was found to vary less than 2-fold between lo-* and M GTPyS (Northup et al., 1982). These findings and other data led to the following proposed mechanism for
’
From the Department of Pharmacology, University of Texas Health Science Center, Dallas, Texas 15235. Receiced March 30, 1984. Supported by National Institutes of Health Grant GM30355 and an Established Investigator Award to E.M.R. from the American Heart Association. *Present address: Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan.
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Abbreviations: G,, stimulatory GTP-binding protein of hormonesensitive adenylate cyclase; GTPyS, guanosine 5’-0-(3-thiotriphosphate); kapp,apparent first-order rate constant for GTPyS binding to G,; Da, dalton; Hepes 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid. A 8000-Da polypeptide also frequently copurifies with G, (Hildebrandt et al., 1984) and probably is a y subunit, analogous to the y subunit of transducin (Fung, 1983). When G, dissociates, the B and y subunits apparently remain associated.
0 1984 American Chemical Societv
5468 B I O C H E M I S T R Y
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the activation of G, (Smigel et al., 1982, 1984) (N is nucleotide): aB
aB-N
X2
Ti-
a
a-N
Either reaction 2 or reaction 4 is the first-order, rate-limiting step. Because kappdoes not vary with the concentration of GTPyS, either the forward rates of reaction 2 and 4 are equal or K,is not in the range of 104-108 M-I. Subunit dissociation, and therefore activation, is promoted by free Mg2+ in the 10-200 mM range. Because the free a subunit is labile to denaturation, the stimulation of dissociation by Mg2+also leads to the loss of total G, activity. Thus, the measured extent of G, activation and GTPyS binding is highly dependent on the nucleotide concentration because the extent of binding reflects the competing reactions of nucleotide binding to the a subunit and that subunit's denaturation (Smigel et al., 1982). This behavior was also observed for G, that had been reconstituted into phospholipid vesicles (Asano et al., 1984). The hormonal regulation of G, activation has been described in much less detail. Some of the best information on the stimulation of G, activation comes from studies of the turkey erythrocyte 0-adrenergic adenylate cyclase system by Levitzki's group [see Tolkovsky (1983) for review]. They stressed that agonist-stimulated activation by nonhydrolyzable GTP analogues is also an apparent first-order process (Tolkovsky et al., 1982) whose rate constant is proportional to the number of agonist-liganded receptors in the membrane (Tolkovsky & Levitzki, 1978). They inferred from these and other kinetic data that the agonist-receptor complex acts formally as a catalyst to promote the activation of multiple molecules of adenylate cyclase at an elevated rate that remains first order. The adenylate cyclase system is quite complex, and its detailed analysis will be vastly simplified by focusing on the interaction of its purified components. Since receptors and G, do not interact functionally in detergent solution, we have reconstituted purified G, and partially purified (1000-4000fold) @-adrenergic receptors with phospholipids to yield a liposomal system that expresses rapid, agonist-stimulated GTPase activity, [35S]GTPySbinding, and G, activation by GTPyS (Brandt et al., 1983; Asano et al., 1984). In this report, we describe the kinetics of the hormone-stimulated activation of G, in these vesicles with respect to the concentrations of agonist and nucleotide. The kinetic pattern is distinctly different from that observed in the Mg2+-promoted activation of soluble G, and suggests a possible sequence of molecular interactions that lead to activation. Experimental Procedures Most of the procedures and the sources of the materials used in this study were reported in the preceding paper (Asano et al., 1984). Receptor-G, vesicles were prepared from p-adrenergic receptors that were partially purified (0.5-2 nmol/mg) from turkey erythrocytes (Brandt et al., 1983), purified rabbit hepatic G, (Sternweis et al., 1981), and a mixture of dimyristoylphosphatidylcholine and turkey erythrocyte polar lipids (Brandt et al., 1983). The molar ratio of receptor to G, in the vesicles used in experiments reported here varied from 3 to 10. GTPyS binding to reconstituted G, was performed as described by Asano et al. (1984). The binding reaction contained 20 mM NaHepes (pH KO), 1 mM EDTA, 0.1 M NaCl, 1 mM
I 0
30
60 TIME ( s e c )
90
J 120
Time courses of isoproterenol-stimulated binding of [35S]GTPySto receptor-G, vesicles at various concentrations of nucleotide. Vesicles were incubated at 30 OC for the indicated times in a binding assay mixture containing 1 mM EDTA, 2 mM MgC12, and various concentrations of [35S]GTPyS,with 1 /.LM(-)-isoproterenol or 0.1 /.LM(-)-propranolol. The incremental amounts of binding due to is0 roterenol are shown as first-orderreplots. Plateau levels of bound [$S]GTPyS were averaged for each concentration of nucleotide to yield Bt and used to normalize binding at shorter times. The data were fit by an unweighted linear least-s uares formula. GTPyS concentrations and the values of Bt were 10-9 (A)(24 fmol), lo-* ( 0 ) (53 fmol), lo-' (m) (82 fmol), and lod M ( r ) (82 fmol). FIGURE 1:
dithiothreitol, 0.1 mM ascorbic acid, and varying amounts of [3SS]GTPyS,isoproterenol, and MgC12 as shown in the text. Binding was assayed at 30 OC, and the binding reaction was initiated by the addition of warmed receptor-G, vesicles to warmed assay medium. The reactions were quenched as described by Asano et al. (1984). The binding of GTPyS to the vesicles was stable in the quenched reaction volumes for several hours at 0 O C . "Basal" binding is defined as that which occurs in the presence of 1 mM free Mg2+. Stimulation of binding by isoproterenol was also measured at this concentration of Mg2+. Stimulation of binding by Mgz+ was achieved with 50 mM MgClZ. Binding rates were generally analyzed according to apparent first-order kinetics. Apparent first-order rate constants, kapp, were obtained as the negative slopes of plots of In [(BT B)/&] vs. time, where BT is the total amount of GTPys bound at long times (after the plateau was reached) and B is the amount of GTPyS bound at individual shorter times. Values of BT were the average of three samples in each experiment, but individual values of B were usually single determinations. Values of kappwere determined from unweighted least-squares fits of these data in the range of [(BT- B ) / B T ]= 0.1-0.9. Reported values of kappare the means from experiments performed by using at least two separate preparations of vesicles. Error bars in the figures indicate the standard deviations. Results The rate of slowly reversible GTPyS binding to G, in reconstituted receptor-G, vesicles was investigated in the presence of various concentrationsof GTPyS, the @-adrenergic agonist isoproterenol, and Mg2+. As an example, Figure 1 demonstrates that, for the agonist-stimulated binding reaction, increasing concentrations of GTPyS increased both the initial rate of binding and the final plateau level of bound ligand. This effect on the plateau level of binding appeared to saturate as would a freely reversible binding equilibrium (Asano et al., 1984). However, the apparent saturation is probably the result of the competition between the binding of ligand to G, and the irreversible denaturation of G, (Smigel et al., 1982; Asano et al., 1984). Because the total concentration of GTPyS was generally much greater than that of G,, binding was analyzed as a possibly pseudo-first-order reaction. First-order replots
VOL. 2 3 , N O . 2 3 , 1984
KINETICS OF HORMONE-STIMULATED GTPyS BINDING
5469
Table I: Effect of the Concentration of GTPyS on MgCl,-Stimulated GTPrS Binding"
10-9 10-8 10-7 10" 3 x 10"
0.26 0.36 0.41 0.33 0.34
(n = 5) ( n = 4) ( n = 4) (n = 4) (mean of 0.23 and 0.45) f 0.06 f 0.05 f 0.13 f 0.05
"The apparent first-order rate constant for GTPyS binding to receptor-G, vesicles was determined in the presence of 50 mM MgCI2 as described in the legend to Figure 2. Data were obtained by using two or three different preparations of vesicles. The error statistic is the standard deviation. The values of kappobserved at 3 X 10" M GTPyS, while similar to those seen at other concentrations, are not highly reliable due to high nonspecific binding.
FIGURE 2: Influence of GTPyS concentrations on the apparent first-order rate constant for the binding of GTPyS to receptor-G, vesicles in the presence or absence of hormone. Apparent first-order rate constants (kapp)for the binding of GTPyS stimulated by 1 WM (-)-isoproterenol ( 0 )were determined from data of the sort shown in Figure 1. Values for kappin the absence of agonist (0)were determined in the presence of 0.1 pM (-)-propranolol. Data are averages from two, three, four, (one point each), or more experiments. The standard deviation of the mean is indicated by brackets where significant. The inset shows kappplotted vs. the logarithm of the GTPyS concentration.
of the binding reactions were linear under all conditions studied. When the G, concentrations were varied by altering the concentration of vesicles in the binding reaction, the rate constants obtained from the slopes of the replots did not change over a 4-fold range of G, concentrations. This was also consistent with a pseudo-first-order reaction. Even at M GTPyS, only 10%of total ligand was bound when the plateau was reached. Thus, the change in rate due to the depletion of free GTPyS would not have been detectable during the course of the reaction, and the binding reaction might have been pseudo first order. The data in Figure 2 describe the dependence of the apparent first-order rate constant for GTPyS binding, kapp,on the concentration of GTPyS. In the absence of agonist (basal rate), kappwas relatively independent of the concentration of GTPyS, suggesting that the binding reaction was simply first order in G,. This behavior of the GTPyS binding reaction was similar to that described for detergent-solubilized G, (Northup et al., 19821, although the rate of binding observed here for reconstituted G, is about 5-10-fold higher than that observed with the soluble protein. This high basal rate was due at least in part to the effect of placing G, in a phospholipid bilayer, since elevated rates were also observed when G, was simply diluted into sonicated dispersions of several different phospholipids. It is also likely that an interaction of G, with the unliganded @-adrenergicreceptor contributes to this high basal rate (S. E. Pedersen and E. M. Ross, unpublished results). In the absence of agonist, the extent of GTPyS binding to reconstituted G, was markedly increased by the addition of MgC12 if the concentration of GTPyS was relatively high (Asano, et al., 1984). This also was the case with soluble G, (Northup et al., 1982). Consequently, the absolute initial rate of binding was also increased by Mg2+. However, the firstorder rate constant for binding was only increased about 2-fold by 50 mM MgC12, and the rate constant measured in the presence of high Mg2+ remained essentially independent of the GTPyS concentration (Table I). The low relative stimulation of the binding rate by 50 mM Mg2+may simply reflect
the fact that the basal rate was already rather high. It is also possible that the concentration of free Mg2+ needed to stimulate the rate of binding to reconstituted G, is either much below 1 mM or much above 50 mM [see Asano et al. (1984)l. In contrast to the basal and Mg2+-stimulatedbinding reactions, the rate of agonist-stimulated GTPyS binding was strongly influenced by the concentration of GTPyS (Figures 1 and 2). Figure 2 shows the effect of the GTPyS concentration in the presence or absence of 1 p M isoproterenol. At 1 nM GTPyS, the agonist-stimulated value of kappwas 0.41 f 0.07 mi&, rather low and only 2-fold greater than the basal k,,, 0.17 f 0.05 mi&. Increasing concentrations of GTPyS increased the isoproterenol-stimulated value of kappto an observed maximum of 4.5 f 0.5 m i d at 1 pM GTPyS. Thus, while the agonist-stimulated binding reaction is clearly dependent on the GTPyS concentration, the nonlinear relationship between kappand the concentration of GTPyS indicates that it is not a simple, second-order bimolecular reaction either (Figure 2). For a bimolecular binding reaction where one reactant (GTPyS) is in excess, the rate should be pseudo first order in the other reactant (G,), but the pseudo-first-order rate constant should be proportional to the concentration of the reactant that is in excess. This is clearly not the case, and kappappears to saturate at high nucleotide concentrations. The agonist-stimulated binding reaction has not yet been studied in detail at higher concentrations of GTPyS because nonspecific binding becomes large and because the high rates are not measurable by manual procedures. However, the data shown here suggest that agonist-stimulated GTPyS binding behaves formally as a reaction that is first order in G, but whose rate constant, kapp,increases as a saturable function of the GTPyS concentration. If the estimated initial rate of binding, rather than kapp,was considered as a function of GTPyS, the basal rate increased in parallel with the plateau amount of GTPyS bound. The data were consistent with the relationship initial rate = IC,&#,. This relationship held for isoproterenol-stimulated binding as well. Because agonist increases Bt (Asano et al., 1984) as well as kaW,the relative stimulation of initial rate caused by agonist was greater than the relative effect on kappshown in Figure 2. The effect of the isoproterenol concentration on the GTPyS-binding reaction was also studied by using similar protocols. Isoproterenol increased the initial rate of GTPyS binding and also increased the maximal amount of ligand that was specifically bound to the vesicles (Asano et al., 1984). The initial rate of GTPyS binding to the receptor-(;, vesicles appeared to be first order at all concentrations of agonist tested. At low nucleotide concentrations, there was little effect of agonist on the first-order rate constant for GTPyS binding,
5470
BIOCHEMISTRY
ASANO AND ROSS 1
1’
[ISOPROTERENOL] (log M)
FIGURE 3: Influence of the isoproterenol concentration on the first-order rate constant for GTPyS binding to receptor-(;, vesicles at various GTPyS concentrations. Binding was measured as described (A),lo-’ (W), or 10” M ( 0 ) in the legend to Figure 1 at either [3SS]GTPyS. The apparent first-order association constant was determined as described in Figure 1. The basal values were determined in the presence of 0.1 WM(-)-propranolol. Data are averages from two experiments (three points), three experiments (five points), four experiments (three points), or more (six points).
in agreement with the data of Figure 2 . As the nucleotide concentration was increased, agonist had a more marked effect on the rate of binding, increasing kappto 2.7 f 0.8 min-I at 100 nM GTPyS (Figure 3). Under these submaximal conditions, the stimulatory effect of isoproterenol appeared to saturate. The agonist produced a half-maximal effect at about 20 nM, well below its equilibrium dissociation constant of 870 nM (Asano et al., 1984). At higher concentrations of GTPyS, isoproterenol caused a 17-fold increase in kappto 5.8 f 2.1. Technical limitations on the speed of the binding assay has limited our ability to measure the maximum possible rate with great accuracy. Because a saturating effect of isoproterenol on kappwas not clearly demonstrated, it is not certain from Figure 3 that kappbehaved as a single saturable function of isoproterenol concentration. However, the data of Figure 3 suggest that, in the presence of 1 pM GTPyS, the half-maximally effective concentration of isoproterenol is at least IO0 nM, about Sfold higher than that observed at the intermediate concentration of nucletide and somewhat closer to the equilibrium Kd for the agonist. Discussion The rate at which GTP binds to and activates G, is a major factor in determining the activity of adenylate cyclase. It is the principal site of hormonal stimulation. The experiments reported here represent our initial kinetic description of the activation process in reconstituted receptor-G, vesicles. In these vesicles, &adrenergic receptors and G, have been substantially purified, the catalytic unit and other receptors have been removed, and the two proteins are present in known and manipulatable concentrations in a predetermined lipid environment. By using GTPyS as the activating ligand, we could study only the activation step itself, since hydrolysis of GTPyS is negligible and reversal of binding is extremely slow. The high-affinity binding of GTPyS to reconstituted G, is a first-order reaction, as is the activation of adenylate cyclase by nonhydrolyzable analogues of GTP in native membranes (Tolkovsky, 1983). The rate, the reaction order, and the regulation of the rate all deserve discussion. The activation of reconstituted G, by GTPyS is considerably faster than that of detergent-solubilized G, under comparable conditions. The basal rate constant reported here, 0.17-0.34 min-I, is at least 5 times greater than that observed for solubilized hepatic G, when stimulated by 10 mM MgClZ (Northup et al., 1982). Likewise, the agonist-stimulated kappdisplayed by reconstituted G, is at least 10-fold greater than the maximum hormonestimulated rate constants that have been observed for the
activation of adenylate cyclase by guanyl-5’-yl imidodiphosphate in membranes from S49 lymphoma cells (Ross et al., 1977), turkey erythrocytes (Tolkovsky & Levitzki, 1978), or rat liver (Birnbaumer et al., 1980a,b) or in a reconstituted system composed of crude G, and receptors from turkey erythrocytes (Gal et al., 1983). The high basal rate and the large hormonal activation that are observed in the reconstituted system can be taken to indicate either highly efficient reconstitution or the nonphysiological nature of the reconstituted vesicles. Clearly, a system that combines proteins from different tissues and different vertebrate classes in synthetic membranes is not, strictly speaking, physiological. However, the highest rates observed here for the activation of G, by GTPyS are no higher than those observed when native membranes are activated by an agonist plus GTP, a rapid process that is usually complete in less than 10 s. The first-order kinetics of G, activation indicates most simply that the rate-limiting step in the process is not the bimolecular association of G, with GTPyS. The activation process that is observed in the vesicles can be explained generally in terms of the model described under the introduction that ascribes the first-order, rate-limiting step to a process closely linked to subunit dissociation (eq 1) (Smigel et al., 1982). We have not directly demonstrated such dissociation during activation of vesicle-bound G,, but the data of Katada et al. (1984) suggest strongly that the subunits of G, do dissociate upon activation in native membranes. The most intriguing aspect of our data is the interaction between agonist and nucleotide in regulating the rate of GTPyS binding. In the absence of agonist, the rate constant for activation was essentially invariant with nucleotide concentration (Table I and Figure 2 ) , as is the case for soluble G, (Northup et al., 1982). In terms of eq 1, this might indicate that rapid GTPyS binding (reaction 3) is obligately preceded by the rate-limiting dissociation step (reaction 2 ) . Alternatively, the aP-N complex may form at higher concentrations of nucleotide, such that the K 1 K4 pathway becomes significant. The aP-N complex would presumably be of low affinity and not detectable by the filtration assay used to measure bound [%]GTPyS or by the reconstitution assay for functional G, activation. If the K , K4 pathway is significant, then the basal and Mgz+-stimulated forward rate constants for reactions 4 and 2 must be virtually identical if kappis to be invariant with the GTPyS concentration. The finding that the rates of activation of G, by GTPyS and either Gpp(NH)p or Gpp(CH,)p are different (Birnbaumer et al., 1980a) supports the K , K4 path. In contrast to basal or Mg2+-stimulated binding, the agonist-stimulated value of kappincreased more than 10-fold with increasing concentrations of GTPyS and appeared to saturate. There was almost no effect of hormone at low concentrations of GTPyS M). It thus appears that increasing concentrations of GTPyS shift kappfrom a low, hormone-insensitive level to a higher, hormone-stimulated level. This suggests that receptor-mediated stimulation of the binding reaction takes place via the K 1 K4 pathway in which the equilibrium of reaction 1 is shifted from a0 to ap-N. The rate-limiting first-order step becomes reaction 4 rather than reaction 2. Reaction 4 appears to be the size of hormonal stimulation. We propose that the agonist-receptor complex acts by accelerating reaction 4 via a transient N-aP-R-H complex (R-H; receptor-hormone). Since R-H can promote the activation of multiple molecules of G, (Asano et al., 1984; Pedersen & Ross, 1982), it evidently behaves formally as a catalyst of this reaction, and R-H need not be assumed to shift
-
-
-
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Biochemistry 1984. 23, 5471-5478 the dissociation equilibrium of the G, subunits. This conclusion agrees with the tendency of R-H to stabilize reconstituted G, and to increase its apparent affinity for nucleotide (Asano et al., 1984). While the mechanism we propose for the hormonal stimulation of G, activation seems to be consistent with our kinetic and quasi-equilibriumdata, it is still clearly speculative. Direct physical measurements of the receptor-G, interaction and the dissociation of the subunits of G, must be made in the vesicles, and the reconstitution of receptor with the resolved subunits of G, will simplify this analysis. Acknowledgments We thank E. A. Lawson for technical assistance and B. Moore for preparation of the manuscript. References Asano, T., Pedersen, S. E., Scott, C. W., & Ross, E. M. (1984) Biochemistry (preceding paper in this issue). Birnbaumer, L., Swartz, T. L., Abramowitz, J., Mintz, P. W., & Iyengar, R. (1980a) J . Biol. Chem. 255, 3542-3551. Birnbaumer, L., Bearer, C. F., & Iyengar, R. (1980b) J. Biol. Chem. 255, 3552-3557. Brandt, D. R., Asano, T., Pedersen, S . E., & Ross, E. M. (1983) Biochemistry 22, 4357-4362. Fung, B. K.-K. (1983) J . Biol. Chem. 258, 10495-10502. Gal, A., Braun, S . , & Levitzki, A. (1983) Eur. J . Biochem. 134, 391-396.
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Hildebrandt, J. D., Codina, J., Risinger, R., & Birnbaumer, L. (1984) J . Biol. Chem. 259, 2039-2042. Katada, T., Northup, J. K., Bokoch, G. M., Ui, M., & Gilman, A. G. (1984) J . Biol. Chem. 259, 3578-3585. Northup, J. K., Smigel, M. D., & Gilman, A. G. (1982) J . Biol. Chem. 257, 11416-1 1423. Northup, J. K., Sternweis, P. C., & Gilman, A. G. (1983) J . Biol. Chem. 258, 11361-11368. Ross, E. M., & Gilman, A. G. (1980) Annu. Rev. Biochem. 49, 533-564. Ross, E. M., Maguire, M. E., Sturgill, T. W., Biltonen, R. L., & Gilman, A. G. (1977) J . Biol. Chem. 252, 5761-5775. Ross, E. M., Howlett, A. C., Ferguson, K. M., & Gilman, A. G. (1978) J . Biol. Chem. 253, 6401-6412. Smigel, M. D., Northup, J. K., & Gilman, A. G. (1982) Recent Prog. Horm. Res. 38, 601-624. Smigel, M. D., Ross, E. M., & Gilman, A. G. (1984) in Cell Membranes, Methods and Reviews (Glaser, L., Frazier, W., & Elson, E., Eds.) Chapter 7, pp 247-294, Plenum Press, New York. Sternweis, P. C., Northup, J. K., Smigel, M. D., & Gilman, A. G. (1981) J . Biol. Chem. 256, 11517-11526. Tolkovsky, A. M. (1983) Curr. Top. Membr. Transp. 18, 11-41. Tolkovsky, A. M., & Levitzki, A. (1978) Biochemistry 17, 3795-38 10. Tolkovsky, A. M., Braun, S., & Levitzki, A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 213-217.
Variation of Transition-State Structure as a Function of the Nucleotide in Reactions Catalyzed by Dehydrogenases. 1. Liver Alcohol Dehydrogenase with Benzyl Alcohol and Yeast Aldehyde Dehydrogenase with Benzaldehyde? Margrit Scharschmidt, Matthew A. Fisher, and W. W. Cleland*
ABSTRACT:
Primary intrinsic deuterium and I3C isotope effects have been determined for liver (LADH) and yeast (YADH) alcohol dehydrogenases with benzyl alcohol as substrate and for yeast aldehyde dehydrogenase (ALDH) with benzaldehyde as substrate. These values have also been determined for LADH as a function of changing nucleotide substrate. As the redox potential of the nucleotide changes from -0.320 V with NAD to -0.258 V with acetylpyridine-NAD, the product of primary and secondary deuterium isotope effects rises from 4 toward 6.5, while the primary I3C isotope effect drops from 1.025 to 1.012, suggesting a trend from a late transition state with NAD to one that is more symmetrical. The values of
Dk (again the product of primary and secondary isotope effects) and I3k for YADH with NAD are 7 and 1.023, suggesting for this very slow reaction a more stretched, and thus symmetrical, transition state. With ALDH and NAD, the primary 13Cisotope effect on the hydride transfer step lies in the range 1.3-1.6%, and the a-secondary deuterium isotope effect on the same step is at least 1.22, but I3C isotope effects on formation of the thiohemiacetal intermediate and on the addition of water to the thio ester intermediate are less than 1%. On the basis of the relatively large I3C isotope effects, we conclude that carbon motion is involved in the hydride transfer steps of dehydrogenase reactions.
V e r y little is known about the structure of the transition states for hydride transfer in enzymatic reactions catalyzed by dehydrogenases, since until recently it was not even possible to determine the intrinsic isotope effects on the hydride transfer step except in rare cases where this step was totally rate limiting (as for formate dehydrogenase; Blanchard & Cleland,
1980). Hermes et al. (1982), however, have developed methods that allow the determination of the intrinsic isotope effects within narrow limits by measurement of I3C isotope effects on C-H cleavage with an unlabeled substrate and one that is deuterated in the primary position. Further, if the I3C isotope effect can be measured with a nucleotide deuterated at the 4-position (this causes an a-secondary deuterium isotope effect), one can obtain an exact solution for all of the intrinsic isotope effects and commitments in the system (Hermes et al., 1982). These techniques allow one to vary the nucleotide substrate and determine the effects on the intrinsic primary
From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706. Received February 13, 1984. This work was supported by a grant from the National Institutes of Health (GM 18938).
0006-2960/84/0423-5471$01SO10
0 1984 American Chemical Society
